Tissues are challenging to genetically manipulate due to limited penetration of viral particles resulting in low transduction efficiency. We are particularly interested in expressing genetically-encoded sensors in ex vivo pancreatic islets to measure glucose-stimulated metabolism, however poor viral penetration biases these measurements to only a subset of cells at the periphery. To increase mass transfer of viral particles, we designed a microfluidic device that holds islets in parallel hydrodynamic traps connected by an expanding by-pass channel. We modeled viral particle flow into the tissue using fluorescently-labelled gold nanoparticles of varying sizes and showed a penetration threshold of only ∼5 nm. To increase this threshold, we used EDTA to transiently reduce cell-cell adhesion and expand intercellular space. Ultimately, a combination of media flow and ETDA treatment significantly increased adenoviral transduction to the core of the islet. As proof-of-principle, we used this protocol to transduce an ER-targeted redox sensitive sensor (eroGFP), and revealed significantly greater ER redox capacity at core islet cells. Overall, these data demonstrate a robust method to enhance transduction efficiency of islets, and potentially other tissues, by using a combination of microfluidic flow and transient tissue expansion.
Fibroblast growth factor receptor-1 (FGFR1) activity at the plasma membrane is tightly controlled by the availability of co-receptors and competing receptor isoforms. We have previously shown that FGFR1 activity in pancreatic beta-cells modulates a wide range of processes, including lipid metabolism, insulin processing, and cell survival. More recently, we have revealed that co-expression of FGFR5, a receptor isoform that lacks a tyrosine-kinase domain, influences FGFR1 responses. We therefore hypothesized that FGFR5 is a co-receptor to FGFR1 that modulates responses to ligands by forming a receptor heterocomplex with FGFR1. We first show here increased FGFR5 expression in the pancreatic islets of nonobese diabetic (NOD) mice and also in mouse and human islets treated with proinflammatory cytokines. Using siRNA knockdown, we further report that FGFR5 and FGFR1 expression improves beta-cell survival. Co-immunoprecipitation and quantitative live-cell imaging to measure the molecular interaction between FGFR5 and FGFR1 revealed that FGFR5 forms a mixture of ligand-independent homodimers (∼25%) and homotrimers (∼75%) at the plasma membrane. Interestingly, co-expressed FGFR5 and FGFR1 formed heterocomplexes with a 2:1 ratio and subsequently responded to FGF2 by forming FGFR5/FGFR1 signaling complexes with a 4:2 ratio. Taken together, our findings identify FGFR5 as a co-receptor that is up-regulated by inflammation and promotes FGFR1-induced survival, insights that reveal a potential target for intervention during beta-cell pathogenesis.
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